Hypoglycemia unawareness and counterregulatory failure are dangerous complications of intensive insulin therapy in type I diabetes. Hypoglycemia-induced glucocorticoid secretion has been implicated in promoting loss of autonomic responses to hypoglycemia. However, glucocorticoids are both counterregulatory hormones themselves and essential for maintaining secretion of epinephrine, one of the most rapid and effective counterregulatory responses. The relative influence of these opposing glucocorticoid actions on defenses against recurrent hypoglycemia are unknown. In addition, glucocorticoids and their primary neural regulator, corticotropin-releasing hormone (CRH), not only influence one another reciprocally but may also have opposing effects on sympathetic tone. Elucidating the mechanisms and impact of glucocorticoid effects on autonomic activity will aid in preventing hypoglycemia unawareness in diabetic patients. To address these issues, this R21 application proposes to (Aim 1) refine a mouse model of hypoglycemia-induced counterregulatory failure, working closely with investigators at the Mouse Metabolic Physiology Core of Vanderbilt University to use hypoglycemic clamp techniques in protocols for recurrent hypoglycemia in mice. Focusing on adrenomedullary epinephrine secretion as a key, glucocorticoid-dependent aspect of counterregulation, we will (Aim 2) use physiological glucocorticoid replacement in CRH knockout mice (CRH KO) to define the relative influence of glucocorticoids vs. CRH on counterregulatory hormone secretion and adrenomedullary activation induced by acute hypoglycemia. We will then (Aim 3) combine the recurrent hypoglycemia procedures defined in Aim 1 with the glucocorticoid manipulations of Aim 2 to test the hypothesis that glucocorticoids inhibit sympathoadrenal responses to recurrent hypoglycemia independently of CRH. Lastly, to identify potential neural mechanisms for the opposing effects of glucocorticoids on epinephrine secretion, we will (Aim 4) map glucocorticoid-dependent changes in expression of marker genes for neuronal activity (c-fos) and specific neurotransmitters in brains of acutely and recurrently hypoglycemic WT and CRH KU mice from Aim 3. These experiments will establish a mouse model for hypoglycemia unawareness, resolve the conflicting effects of glucocorticoids on sympathoadrenal activity, and reveal potential mechanisms for hypoglycemia-induced counterregulatory failure.